Aspergillus fumigatus is the causative agent for medical conditions including invasive aspergillosis, which is a major cause of morbidity and mortality in immunocompromised patients. The survival of such patients depends on early diagnosis and prompt initiation of effective antifungal treatment (see Latge, 1999, Aspergillus fumigatus and spergillosis. Clinical Microbiology Reviews 12:310-350; and Man et al., 2002, Aspergillosis. Pathogenesis, clinical manifestations, and therapy. Infectious Disease Clinics of North America 16:875-894). Azole-based compounds (e.g., triazoles) are the most commonly used antifungal drugs. The target for these compounds is the Cyp51A protein. This protein is involved in the synthesis of ergosterol, which is a bulk sterol component of fungal cell membranes (see Vanden Bossche, 1985, Biochemical targets for antifungal azole derivatives: hypothesis on the mode of action. Current Topics in Medical Mycology 1:313-351).
Unfortunately, conventional laboratory tests for the presence of Aspergillus fumigatus, such as culture and galactomannan detection, lack sensitivity, and are rarely conclusive, resulting in true positive results only at advanced stages of infection or necessitating invasive procedures for formal microbiological evaluation (see Denning, 1998, Invasive aspergillosis. Clinical Infectious Diseases 26:781-803; and Latge, 1999, Aspergillus fumigatus and aspergillosis. Clinical Microbiology Reviews 12:310-350). Furthermore, the emergence of clinical resistance to azole-based drugs impedes successful treatment of infection by Aspergillus fumigatus (see Denning et al., 1997, Itraconazole resistance in Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy 41:1364-1368; Mar et al., 2002, Aspergillosis, Pathogenesis, clinical manifestations, and therapy. Infectious Disease Clinics of North America 16:875-894; and Steinbach et al., 2003, Review of newer antifungal and immunomodulatory strategies for invasive aspergillosis. Clinical Infectious Diseases 37 Supplement 3:S157-S187).
For example, isolates of Aspergillus fumigatus exhibiting increased tolerance to triazoles, relative to wild-type Aspergillus fumigatus, have been identified. Each of these isolates was found to have a mutation in cyp51A responsible for an amino acid substitution at position 54, 138, 220, or 448 of the Cyp51A protein, wherein each of these amino acid substitutions gave rise to the reduced triazole susceptibility. (see Mann et al., 2003, Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14 alpha-demethylase. Antimicrobial Agents and Chemotherapy 47:577-581; Nascimento et al., 2003, Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole. Antimicrobial Agents and Chemotherapy 47:1719-1726; Diaz-Guerra et al., 2003, A point mutation in the 14 alpha-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy 47:1120-1124; Xiao et al., 2004, Three-dimensional models of wild-type and mutated forms of cytochrome P450 14 alpha-sterol demethylases from Aspergillus fumigatus and Candida albicans provide insights into posaconazole binding. Antimicrobial Agents and Chemotherapy 48:568-574; and Mellado et al., 2004, Substitutions at methionine 220 in the 14 alpha-sterol demethylase (Cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. Antimicrobial Agents and Chemotherapy 48:2747-2750).
Thus, there exists a critical need to develop techniques to facilitate the early and reliable diagnosis of invasive aspergillosis by detecting the causative agent Aspergillus fumigatus in an infected patient, and to ascertain whether treating this patient with a triazole would be effective by determining whether the infecting Aspergillus fumigatus encodes a Cyp51A protein having one or more of the above-noted amino acid substitutions.